Carbodiimides
are a group of organic compounds which have the resonance formula
N=C=N. Carbodiimide is formed by dehydration of urea or from thiourea.
Carbodiimides are readily reacts with various form of amines and
hydroxyl functional
groups. Carbodiimides are used as dehydration agents and
as activating agent of carboxylic acids to form esters or amides. The disubstituted
carboxyl activating agents are used for crosslinking
proteins to nucleic acids and formation of immunoconjugates
in peptide synthesis. N,N'-dicyclohexylcarbodiimide (DCC)
is one of the most
common peptide coupling agents. It is a low melting point waxy solid; insoluble
in water but highly soluble in common organic solvents
like acetonitrile dichloromethane, dimethylformamide,
and tetrahydrofuran. DCC is a potent allergen. N,N'-diisopropylcarbodiimide
(DIC) is an alternative to DCC, DIC is a liquid and
is not an allergen. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) is a water-soluble
activating agent for amide bonding with primary amines. It also activates phosphate
groups. Typically, it is utilized in the pH
range 4.0-6.0 without buffers. In particular, amine and carboxylate buffers
should be avoided. Carbodiimides
are so active and cause racemization of the
amino acid. Active esters are
less reactive and less in danger of racemization. 1-Hydroxybenzotriazole (HOBt) and
1-hydroxy-7-aza-benzotriazole (HOAt)
are substances that react with the O-acylurea to form active esters.
HOAt is a condensation
additive in peptide synthesis. It
efficiently speeds up coupling process,
reduces the loss of chiral integrity, and
provides a visual indication (yellow to
colorless) of the reaction course. Other
active esters exit as non-nucleophilic anionic salts
of uronium or phosphonium
such as O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate(HBTU), O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP).

PyBOP
is a coupling reagent for solid-phase peptide synthesis without
forming carcinogenic HMPA as by-product.